A thorough understanding of DNA replication is likely to require a detailed characterization of the DNA sequences and proteins which regulate the initiatin process. Because the replication mechanism of the autonomous pravoviruses is similar to that proposed for telomere replication in eukaryotes and because the replication of parvovirus DNA is highly dependent on host factors expressed during S phase, these viruses provide an excellent model for studying control of DNA replication. The murine parvovirus, Minute Virus of Mice (MVM), will be used to study steps in the intiation of viral DNA replication. The replicative form DNA of MVM has been cloned and when the uncut cloned DNAS is transfected into mouse fibroblasts, the MVM DNA is specifically exciesd, replicated, and gives rise to infectious virions. The MVM DNA hs a 60Kd terminal protein covalently bound to the 5' termini which is believed to be involved in initiation of MVM DNA replication by acting as a site specific nicking-closing enzyme. Preliminary data with an antibody raised to the terminal protein suggest that it is a host gene product. The nature of the MVM DNA replication origin will be studied to determine the DNA sequences required to initiate MVM DNA replication and to determine the role of the terminal protein in this process. Specific problems to be addressed during this grant are as follows: 1. Identification of DNA sequences required for initiation of MVM DNA replication by analysis of viability and ability to replicate of a series of deletion mutants (constructed by Bal 31 section) in the MVM replication origin. 2. Confirmation that the MVM terminal protein is host encoded - by Western blot analysis of lysates from infected and uninfected cells using an antibody to MVM terminal protein. 3. Identification of cellular DNA sequences bound by the MVM terminal protein -by antibody selection of terminal protein bound fragments of Protein-A Sepharose followed by cloning and sequencing of the selected cellular DNA fragments.